Construction and Cloning of Human Grenulocyte-Colony Stimulating Factor (hG-CSF) cDNA

Human granulocyte colony-stimulating factor (hG-CSF) is a growth factor that stimulates the proliferation and differentiation of granulocyte progenitor cells, and neutrophilic granulocyte colony formation of bone marrow cells. This factor also induces terminal differentiation of some leukemic myeloid cells. HuG-CSF is produced in human monocyte and macrophage in response to bacterial endotoxin, and also in human cell lines such as oral cavity carcinoma (CHU-2) and bladder carcinoma (5637). The aim of this research was to extract total RNA from stimulated human monocyte cells, synthesis of hG-CSF ds cDNA, and cloning ofcDNA in an appropriate cloning vector. First, monocyte cells were isolated from normal human peripheral blood.These cells were cultured and simultaneously stimulated by LPS and hIFN-y. Total RNA was extracted from cells, and hG-CSF signal sequence-containing cDNA and signal sequence-free cDNA were synthesized with specific primers and based on RT-PCR method. Also, both of the mentioned cDNAs were sythesized by extracting total RNA from cultured 5637 carcinoma cells. Both of the monocytic cDNAs were confirmed by the restriction enzyme of Styl that forms three distinct fragments on gel electroforesis. Then, signal sequence-containing cDNA was inserted into the cloning vector of pBluescriptIISK and cloned in E.coli (TOPIOF' strain). After screening, correct colonies were selected, and recombinant vector was extracted from transformed cells and confirmed by the restriction enzymes of EcoRl, Baml-ll, Ncol and Styl, Finally, the correct recombinant vector was selected and sequenced. After confirmation of the nucleotide sequence of the cloned cDNA, the signal sequence-free cDNA was synthesized with specific primers, recombinant vector as a templet, and by PCR; and was inserted into pBluescriptIISK and cloned in TOPIOF'. Then, the correct clone was selected and confirmed by restriction enzymes.